The invention relates enhanced Agrobacterium transformation frequencies of plants due to overexpression of the histone H2A encoded by the Arabidopsis RAT5 gene. At present, many Arabidopsis ecotypes and mutants cannot be easily or efficiently transformed by a root transformation method, generally using Agrobacterium. Agrobacterium tumefaciens is a gram negative soil bacterium that has been exploited by plant biologists to introduce foreign DNA into plants. Although known for this practical application, the actual mechanism of DNA transfer from bacteria to plants is not completely understood. Moreover, there are some limitations on the use of this transforming vector, e.g. difficulties in transforming monocots, and transforming frequencies may be too low to be useful.
Agrobacterium tumefaciens genetically transforms plant cells by transferring a portion of the bacterial Ti-plasmid, designated the T-DNA, to the plant, and integrating the T-DNA into the plant genome. Little is known about the T-DNA integration process, and no plant genes involved in integration have previously been identified. The DNA that is transferred from Agrobacterium to the plant cell is a segment of the Ti, or tumor inducing, plasmid called the T-DNA (transferred DNA). Virulence (vir) genes responsible for T-DNA processing and transfer are reported to lie elsewhere on the Ti plasmid. The role of vir genes in T-DNA processing, the formation of bacterial channels for export of T-DNA, and the attachment of bacteria to the plant cell are reported (Sheng and Citovsky, 1996; Zupan and Zambryski, 1997). In contrast, little is known about the role of plant factors in T-DNA transfer and integration.
An international patent application (WO97/12046) describes how to improve integration of exogenous DNA by delivering the DNA into plant cells with one or more Agrobacterium genes that can encode for proteins within the plant cells. This technique, referred to as “agrolistic transformation” (p. 35) is just an improvement over biolistic transformation by which DNA is delivered to the plants by a non-biological method. In this technique, genes encoding virulence proteins that normally function in Agrobacterium are transferred to the plants along with a T-DNA substrate. The substrate is then acted upon in the plant cell to make a T-DNA molecule. The technique described does not include the use of plant genes, or of histones at all, and does not utilize histones to increase either integration of T-DNA or transformation frequency. The technique was not shown to make a plant more susceptible to transformation. The goal of WO97/12046 was to increase predictability of the location of integration, not its frequency. “Agrolistic transformation” is an expensive procedure requiring much infrastructure and resources; one of skill has to go through the laborious process every time to develop a transgenic plant.
The isolation of a putative plant factor has recently been reported. Ballas and Citovsky showed that a plant karyopherin α (AtKAP α) can interact with VirD2 nuclear localization sequences in a yeast two-hybrid interaction system, and is presumably involved in nuclear translocation of the T-complex. Using a similar approach, a tomato type 2C protein phosphatase, DIG3, that can interact with the VirD2 NLS was identified. Unlike AtKAP α, DIG3 plays a negative role in nuclear import. After the T-DNA/T-complex enters the nucleus, it must integrate into the plant chromosome. Plant chromosomal DNA is packaged into nucleosomes consisting primarily of histone proteins. The incoming T-DNA may have to interact with this nucleosome structure during the integration process. However, T-DNA may preferentially integrate into transcribed regions of the genome. These regions are believed to be temporarily free of histones. How exactly T-DNA integration takes place is unknown. Recent reports have implicated involvement of VirD2 protein in the T-DNA integration process. Plant proteins are also likely to be involved in this process (Deng et al., 1998; Ballas and Citovsky, 1997). Other suggestions for the involvement of plant factors in T-DNA transfer and integration comes from identification of several ecotypes of Arabidopsis that are resistant to Agrobacterium transformation.
Transforming the transformation resistant rat5 mutant with a wild-type RAT5 (histone H2A) gene was reported by the inventors to complement the mutant phenotype.
There are several steps in which plant genes are likely involved in the Agrobacterium-mediated transformation process. First, plant-encoded factors could be involved in the initial step of bacterial attachment to the plant cell surface. Mutants and ecotypes that are deficient in bacterial attachment have been identified, and genes involved in bacterial attachment are currently being characterized. The next step in which a plant factor(s) could be involved is the transfer of T-strands from the bacteria to plant cells across the plant cell wall and membrane. After the T-DNA/T-complex enters the cytoplasm of the plant cell, plant factors are required to transport the T-complex to the nucleus.
T-DNA integration does not appear to take place by homologous recombination, believed to be the most common method of foreign DNA integration in prokaryotes and lower eukaryotes, because no extensive homology between the T-DNA and target sequences has been found. T-DNA is reported to integrate by illegitimate recombination (Matsumoto et al., 1990; Gheysen et al., 1991; Mayerhofer et al., 1991; Ohba et al., 1995). Illegitimate recombination is the predominant mechanism of DNA integration into the genomes of higher plants (Britt, 1996; Offringa et al., 1990; Paszkowski et al, 1988).
Information on factors affecting Agrobacterium transformation frequencies in plants is needed to improve performance of this method.